in B cell receptor signaling
Dissertation
for the award of the degree
”Doctor rerum naturalium”
of the Georg-August-Universit¨ at G¨ ottingen
within the doctoral program Molecular Biology of Cells of the Georg-August University School of Science (GAUSS)
submitted by Julius K¨ uhn
from Frankfurt am Main
G¨ ottingen 2015
Prof. Dr. J¨ urgen Wienands, Department of Cellular and Molecular Immunology, University Medical Center G¨ ottingen
Prof. Dr. Blanche Schwappach, Department of Molecular Biology, University Medical Center G¨ ottingen
Prof. Dr. Henning Urlaub, Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry
Members of the Examination Board
Referee: Prof. Dr. J¨ urgen Wienands, Department of Cellular and Molecular Immunology, University Medical Center G¨ ottingen
2
ndReferee: Prof. Dr. Blanche Schwappach, Department of Molecular Biology, University Medical Center G¨ ottingen
Further members of the Examination Board
Prof. Dr. Henning Urlaub, Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry
Prof. Dr. Michael Thumm, Department of Cellular Biochemistry, University Medical Center G¨ ottingen
Prof. Dr. Dieter Kube, Department for Hematology and Oncology, University Medical Center G¨ ottingen
Prof. Dr. Uwe Groß, Department of Medical Microbiology, University Medical Center G¨ ottingen
Date of oral examination: 28
thof May 2015
Herewith I declare that I prepared the doctoral thesis ”Multimolecular adaptor protein complexes in B cell receptor signaling” on my own with no other sources and aids than indicated.
G¨ ottingen, 13
thof April 2015
Julius K¨ uhn
1 Summary 1
2 Introduction 2
2.1 Discovery of antibodies and B cells: a story of 130 years . . . 2
2.2 Current understanding of BCR structure and signaling . . . 3
2.2.1 Structure of antibodies and BCR . . . 3
2.2.2 Antibody function . . . 5
2.2.3 Activation of BCR signaling by antigen binding: still partially a mystery . . . 6
2.3 SLP65: A central adaptor molecule for BCR signaling . . . 7
2.4 The SLP65 regions and their functions . . . 9
2.5 CIN85: a multi-domain adaptor protein with still mysterious functions . . . 11
2.6 Aim of this study . . . 14
3 Material and methods 15 3.1 Material . . . 15
3.1.1 Instruments . . . 15
3.1.2 Software . . . 16
3.1.3 Consumables . . . 17
3.1.4 Chemicals and reagents . . . 18
3.1.5 Buffers and solutions . . . 20
3.1.6 Antibodies . . . 21
3.1.7 Enzymes . . . 22
3.1.8 Media . . . 22
3.1.9 PCR Primers . . . 23
3.1.10 Plasmids . . . 25
3.1.11 Bacteria . . . 31
3.1.12 Eukaryotic cell lines . . . 31
3.2 Methods . . . 32
3.2.1 Genetic methods . . . 32
3.2.1.1 Polymerase chain reaction (PCR) . . . 32
3.2.1.2 Overlap extension PCR . . . 32
3.2.1.3 Site directed mutagenesis . . . 33
3.2.1.4 Enzymatic digest of DNA . . . 33
3.2.1.5 Agarose gel electrophoresis . . . 33
3.2.1.6 Purification of DNA from agarose gels or PCR reactions . . . 34
3.2.2 Cell biological methods . . . 35
3.2.2.1 Culturing of eukaryotic cells . . . 35
3.2.2.2 Freezing and thawing of eukaryotic cells . . . 35
3.2.2.3 SILAC labeling . . . 35
3.2.2.4 Retroviral transduction . . . 35
3.2.2.5 Transfection of eukaryotic B cells by Electroporation . . . 36
3.2.3 Biochemical methods . . . 36
3.2.3.1 Preparation of cell lysates . . . 36
3.2.3.2 Affinity purification . . . 36
3.2.3.3 VAMP7+ vesicle enrichment . . . 37
3.2.3.4 Co-immunoprecipitation . . . 37
3.2.3.5 Subcellular fractionation by Balch homogenization . . . 37
3.2.3.6 Subcellular fractionation by cavitation . . . 38
3.2.3.7 SDS-PAGE . . . 38
3.2.3.8 Coomassie staining . . . 38
3.2.3.9 Immunoblotting . . . 39
3.2.3.10 Recombinant protein production inE. coli . . . 39
3.2.4 Optical methods . . . 39
3.2.4.1 Confocal microscopy . . . 39
3.2.4.2 Flow Cytometry . . . 40
4 Results 41 4.1 CIN85 reduces the threshold for BCR recruitment of SLP65. . . 41
4.2 A fraction of CIN85 and SLP65 is membrane associated in resting B cells. . . 41
4.3 VAMP7+ vesicles display lysosomal characteristics but no detectable association with CIN85 and SLP65. . . 46
4.4 Plasma membrane anchoring of SLP65 cannot substitute for the interaction with CIN85 . 46 4.5 The CIN85-CC supports SLP65 function. . . 48
4.6 The CIN85-CC is an autonomous domain and can be assembled from its two halves. . . . 52
4.7 The CIN85-CC and the SLP65 SH2 domain have distinct functions. . . 55
4.8 The interactome of CIN85 is diverse, but the number of interaction partners of the CIN85- CC is diminutive. . . 55
4.9 The CC mediates PA association, but this is not sufficient to support SLP65 function. . . 63
4.11 An amino acid exchange in the hydrophobic interface of the CC disrupts its oligomerization
and function. . . 66
4.12 Other CC domains can replace the CIN85-CC to support SLP65. . . 69
4.13 Oligomerization enables SLP76 to take part in the BCR signaling cascade. . . 75
4.14 Oligomerization of SLP65 is sufficient to enable BCR recruitment and Ca2+ signaling. . . 75
5 Discussion 80 5.1 CIN85-mediated oligomerization of SLP65 . . . 80
5.2 The formation of the SLP65-CIN85 complex . . . 82
5.3 Inhibiton of complex formation by intramolecular CIN85 interactions . . . 83
5.4 The influence of SLP65 and CIN85-CC lipid binding . . . 84
5.5 The chronological order of SLP65-CIN85 complex formation . . . 85
5.6 The regulation of oligomerization as a general feature in signaling pathways . . . 87
5.7 The transport of SLP65 and CIN85 to the plasma membrane . . . 89
5.8 The overall effect of CIN85 expression in B cells . . . 91
5.9 Conclusion . . . 94
6 Bibliography 95
7 Appendix I
7.1 The Proteome of VAMP7+ vesicles in DT40 B cells . . . I 7.2 Amino acid single letter code . . . VI 7.3 Deoxyribonucleotide single letter code . . . VI 7.4 Abbreviations . . . VII 7.5 Figures . . . XIII 7.6 Tables . . . XIV
8 Acknowledgements XV
The activation of B cells is the crucial step of the humoral immune response. This process is controlled by the B cell receptor (BCR) signaling pathway which is initiated by antigen recognition. An elaborated and detailed understanding of this pathway is of great interest given that its misregulation is involved in the genesis of leukemia and immunodeficiencies. The adaptor protein SLP65 is a key regulator in BCR signaling. After phosphorylation of its tyrosine residues, SLP65 serves as assembly site for enzymes and facilitates their mutual activation. The activation of SLP65 by phosphorylation is a multi-step process that requires SH3 domain-binding proline motifs within SLP65. These motifs mediate the interaction of SLP65 with the adaptor protein CIN85. While it was known at the beginning of my doctoral thesis project that the SLP65-CIN85 interaction supports the function of SLP65 in the BCR pathway, the un- derlying mechanism remained totally unclear. The elucidation of this mechanism constitutes the aim of my doctoral thesis.
By a genetic approach including chimeric proteins I could localize the effector function of CIN85 in its C-terminal coiled-coil (CC) domain. To get insight into the function of the CIN85-CC, I determined the protein interactome of CIN85 by SILAC-based mass spectrometry and investigated membrane lipid binding by biochemical means. Next, I focused on the structure of the CIN85-CC in cooperation with Prof. C. Griesinger’s group. By NMR-spectroscopy, we obtained the result that the CIN85-CC forms a stable trimer. In further genetic experiments, I determined that oligomerization of SLP65 is essential for its function in the BCR signaling pathway. Because CIN85 forms a trimer with nine SLP65 binding sites, it provides the required oligomerization by simultaneous binding of several SLP65 molecules . Fur- thermore, I found out that the SLP65 N-terminus is a pre-requisite for efficient SLP65 oligomerization.
As the N-terminus targets SLP65 to intracellular membranes, I could identify vesicles as the platform for SLP65-oligomerization by CIN85. Because CIN85 and its homolog CD2AP interact with several other proteins in the same manner as with SLP65, this mechanism is likely a general biological principle of action for the CIN85-family adaptor proteins.
2 Introduction
2.1 Discovery of antibodies and B cells: a story of 130 years
The immune system is the crucial line of defense of every multicellular organism against pathogens and cancer development. Antibodies are soluble proteins that recognize and fight pathogens specifically. They play a key role for an effective immune response. The importance of molecules with antibody-like function can be seen from the fact that they are found in all vertebrates from lamprey on (reviewed in [28]).
The discovery of antibodies is closely linked to the discovery of the immune system itself. The first scientific approach to understand the immunizing factors of our body were made in the ending 18th century. Edward Jenner discovered that a human being was protected from smallpox infection after the application of the relatively harmless cowpox virus (vaccinia) (reviewed in [193]). This was the birth of the procedure nowadays known as vaccination.
The key discoveries for the evolvement of immunology as a science were made in the second half of the 19th and the beginning of the 20th century (reviewed in [22] and [156]). They are linked to names like Louis Pasteur (first rabies vaccination), Emil von Behring, Paul Ehrlich and Ilja Metchnikow. Two fundamental concepts were developed in this time: There was the finding of Behring and Kitasato that diphtheria-infected children could be cured by the application of the serum of diphtheria-infected rabbits (serotherapy). The phenomenon was calledhumoral immunity, pathogen resistance transferred by liquid (humor in Latin). This concept was postulated, amongst others, by P. Ehrlich, who proposed the ”side chain theory”. Receptors (”side chains”) on the cell surface would recognize a pathogenic structure which would lead to the secretion of soluble components that have the ability to neutralize pathogens. The other concept, strongly promoted by I. Metchnikow and others, based on phagocytosis (Ancient Greek for ”devouring by a cell”). It was observed that specific cells can internalize, phagocytose, pathogens like bacteria. The process of phagocytosis was termed cellular immunity because it was conferred by cells, not by serum. The two concepts of humoral and cellular immunity were initially seen contradictory. The insight that both of them can be unified developed only later on.
The components of the humoral immune response, i.e. the serum molecules that could transfer immunity between individuals, remained enigmatic for a long time. The molecules we know today as ”antibodies”, a term used by P. Ehrlich already in 1891, were also called ”fixatives” or ”sensitizing substances” (I.
Metchnikow) at the beginning. Their astonishing abilities to cause blood agglutination, bacterial lysis, and systemic protection of the organism at the same time led to confusion about their nature and ori- gin. Until the 1940s, it was widely believed that phagocytotic cells were also responsible for antibody production. This picture was challenged when M. Bjørneboe and H. Gormsen described the production of antibodies by plasma cells and proposed that lymphocytes and plasma cells could be involved in the generation of antibodies. The antibody production by plasma cells was later confirmed by experiments
Jerne and A. Nordin. The question of the origin of plasma cells was still open until the 1960s, when it could be shown by J. Gowans that plasma cells originate from lymphocytes which led to a conclusive theory of the cellular source of antibodies.
Lymphocytes, already discovered in the late 19thcentury by A. Maximow, P. Ehrlich and others, could not be assigned to an exact immunological function for a long time period. J. Murphy succeeded in showing their importance for the immune response already in the 1910s, but their mechanistic role re- mained completely unknown. In 1965, M. Cooper could classify lymphocytes according to the origin of their development in T-lymphocytes (development in theThymus) and B-lymphocytes (development in the Bursa fabricii in chicken, in the bone marrow in mammals) (reviewed in [27]). His work could also identify B-lymphocytes to be responsible for antibody production. T-lymphocytes have been shown to play an essential role for cellular immunity and possess a supportive role in B-lymphocyte-mediated production of antibodies.
Having established the cellular framework of antibody production, it was possible to investigate the molecular mechanisms in detail. This was especially facilitated by the development of new biochemical and genetic methods, most notably protein sequencing and polymerase chain reaction. One burning question that had occurred in this time was how the immense repertoire of diverse antibodies against virtually every pathogenic structure was generated. Germ-line coding of a myriad of different antibodies would overextend the limits of the human genome. The model of DNA recombination was developed in the 1970s by S. Tonegawa and others to explain this phenomenon (see section 2.2.1 for details).
The new methods enabled the identification of proteins and genes that are necessary for B cell function.
The recognition tool of B cells for their specific pathogen-derived target structure, the antigen, was iden- tified to be a membrane-associated antibody, termed B cell receptor (BCR). This sensing of the antigen presence is essential for the activation of a B cell and hence the whole humoral immune response. The details of molecular processes involved in BCR-signal propagation have been studied since at least three decades, yet there remain riddles and open questions that are still waiting to be solved.
2.2 Current understanding of BCR structure and signaling
2.2.1 Structure of antibodies and BCR
The BCR consist of an antibody molecule containing a transmembrane domain and the two associated signaling chains Igα(CD79a) and Igβ (CD79b) [21, 146]. The Igα-Igβ heterodimer associates with the membrane-anchored antibody by non-covalent interactions [21, 187, 139]. In general, the BCR allows the B cell to detect the presence of its antigen by antigen binding of the antibody part and subsequent trans- duction of this signal by the signaling chains. The molecular structure of an antibody, or immunoglobulin
(Ig), molecule was elucidated in the 1960, mainly by works of R. Porter and G. Edelman (reviewed in [188] and [162]). An immunoglobulin molecule consists of two Ig heavy chains and two Ig light chains.
They form a Y-shaped ensemble with individual chains interlinked by disulfide bridges. The two heavy chains are linked by a varying number of disulfide bridges, while each light chain is linked to one heavy chain by usually one disulfide bridge [101]. The antibody molecule can be separated in two functionally distinct regions: the variable (V) domains which is responsible for antigen binding and the constant (C) domains which exhibits the effector functions of the antibody. Each chain of the antibody consists of several immunoglobulin super-family (IgSF) domains. IgSF domains display a characteristic fold of two sandwichedβ-sheets, linked by one disulfide bridge. The N-terminal IgSF domain of each Ig chain is the V domain. The following C-terminal IgSF domains are C domains. Ig light chains contain only one C domain, while Ig heavy chains contain three or four C domains, depending on the antibody class. The first (CH1) and the second (CH2) domain surround a spacer region which forms a ”hinge”, resulting in the Y-shaped structure of an antibody shown in Figure 2.1.
Fig. 2.1: Schematic structure of an antibody molecule. An antibody consists of two Ig light and to Ig heavy chains. The chains are linked by disulfide bridges. The structure of an antibody resembles an ”Y”
due to the hinge region between the C domains CH1 and CH2. The antigen binding sites are located within the V domains, the N-terminal IgSF domain of each chain. The following IgSG domains, the C domains (three for IgG), provide binding sites for Fc receptors and complement components.
The entire repertoire of immunoglobulins has the ability to bind to a nearly all occuring antigenic pat- terns, despite the structural heterogeneity of the antigens. This variety is enabled by genomic DNA- recombination of the multigene family coding Ig heavy and light chains. The V domain is encoded by V, D, and J gene segments, while the C domains are encoded by whole exons. The V, D, and J segments are arranged by DNA-recombination to constitute a complete V domain. Because the genome contains
Imprecise segment arrangement, the introduction of single point mutations, and the presence of two different Ig light chains, termed κand λ, can further enlarge the antibody repertoire. Altogether, the number of potentially produced immunoglobulin molecules can be estimated to exceed 1016. The really observed repertoire of antibodies and BCRs is nevertheless more limited. B cells undergo positive and negative selection processes during their development (reviewed in [37]). Furthermore, the fate of a B cell depends on the presence of its antigen. Generally, B cells that encounter their antigen start to proliferate.
Thereby they can out-compete other B cells, a process termed clonal selection by F. M. Burnet (reviewed in [140]).
In contrast to the huge diversity of the V domains, the C domains of an antibody are germ-line encoded without further modifications. The C domains of an Ig heavy chain can originate from nine different CH genes, defining the human antibody classes. The CH region of each class can be linked to an arranged V domain by DNA recombination in a process termed class switch recombination. The CH region undergoes alternative splicing so that the same transcript can encode for a soluble antibody or a membrane bound BCR.
2.2.2 Antibody function
In humans, there exist the five antibody classes IgA (subclasses IgA1 and IgA2), IgD, IgE, IgG (sub- classes IgG1-IgG4) and IgM. They differ in their structure and as a result also in their biologic functions.
The effector functions of an antibody are determined by the C-terminal IgSF-domains of its Ig heavy chain. The domains CH2-CH3 (for IgG, IgD and IgA) or CH2-CH4 (for IgM and IgE) represent the Fc (Fragment crystallizable) part of an antibody. The Fc part mediates the antibody effector function because it provides the ability to cause antibody-dependent cellular cytotoxicity (ADCC) or complement- dependent cytotoxicity (CDC). ADCC is mediated by Fc receptors (FcRs) that bind to the Fc part of an antibody. Many cells of the immune system, e.g. monocytes and macrophages, display Fc receptors on their surface. This allows them to recognize antibody-coated structures like a pathogen cell and attack them by phagocytosis or lysis due to cytotoxin secretion. By means of CDC, antibodies can target cells for destruction by the complement system. The complement component C1q has the ability to bind to Fc parts and to initiate the lysis of the antibody coated cell. Both processes, ADCC and CDC, depended on the glycosylation pattern of the Fc part. Besides these effector functions, antibodies can also directly neutralize their antigen. They can e.g. inhibit the cell entry of a pathogen by masking a protein structure involved in this process or prevent the effect of toxins.
The membrane-bound form of IgM is the first BCR expressed in the B cell development and it is found on all na¨ıve B cells. Analogously, the secreted form of IgM is the first immunoglobulin produced in an immune response. It forms a pentamer that has a high ability to work by CDC. As IgM antibodies
originate normally from cells that did not experience extended affinity maturation in a germinal center reaction, their affinity towards the antigen is low compared to other antibody classes. The pentameric nature of IgM can compensate for this by an increased avidity.
The IgD-BCR is expressed on na¨ıve B cells and originates from the same mRNA as the IgM-BCR by alternative splicing. The serum levels of soluble IgD are extremely low. Its physiological function is not yet clarified.
Monomeric IgG antibodies display the main portion of immunoglobulins in the serum. Because IgG originates from class switching during a germinal center reaction, IgG antibodies have usually undergone affinity maturation and possess a high affinity towards their antigen. This makes them efficient neutral- izers for toxins and viruses. The Fc part of IgG binds to a variety of FcγRs present on many cell types of the immune system, thus IgG mediates ADCC most efficiently. In comparison to the IgM- and IgD-BCR, the IgG-BCR has an additional cytoplasmatic motif, the immunoreceptor tail tyrosine (ITT) motif that contributes to BCR signaling in addition to Igαand Igβ [41].
IgA antibodies are the most abundant immunoglobulins at mucosal surfaces. They are mainly present as monomers in the serum, but as dimers in the mucosa. IgA can act through neutralization of antigens, preventing the uptake of pathogens at mucosal surfaces. It can also cause ADCC by binding to the FcαR which is present e.g. on neutrophiles.
The level of soluble IgE antibodies is very low. They are implicated in the defense against parasites and the triggering of allergies. IgE binds to the FcR on mast cells with an extraordinary high affinity. Like the IgG-BCR, the IgE-BCR possesses an ITT motif [41].
2.2.3 Activation of BCR signaling by antigen binding: still partially a mystery
Despite the structure of the BCR molecule is known in very detail, the oligomerization state of the BCR in resting cells is still under debate (reviewed in [134]) . It has been found that the BCR is mainly mobile and monomeric without stimulation by antigen binding [183]. Contradictory, it was reported that the BCR is present on resting cells as a signaling inactive dimer or higher oligomer [198]. Also the change of the oligomerization state after antigen binding is seen controversially. According to the most established model, signaling-inactive BCR monomers are physically crosslinked by encountering their antigen which leads in turn to the formation of oligomeric signaling-active BCR complexes, described as microclusters (reviewed in [134]). On the other hand, the dissociation activation model proposes the presence of signaling-inactive oligomers which would dissociate after antigen encounter, forming signaling active monomers [198, 85].
Regardless which model of BCR activation might fit best to reality, the next essential step after the antigen encounter is the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) in the BCR-signaling chains Igαand Igβ. One ITAM consist of the sequence YXXI/LX7YXXI/L that
and the spleen tyrosine kinase (Syk) have been found to be implicated in ITAM phosphorylation [32, 85].
The exact triggering of the ITAM phosphorylation process remains partially unclear. Changes in BCR conformation as well as a changed lipid environment of BCR oligomers seem to play a role (reviewed in [134]). The tyrosine phosphorylation of the ITAMs creates binding sites for the tandem SH2 domains of Syk [191, 45]. The interaction of Syk with the phosphorylated ITAMs leads to an enhancement of the Syk kinase activity [150]. This activation of Syk is essential for BCR signaling because Syk phosphorylates the adaptor protein SLP65, also called BLNK [47]. The complex of the two adaptor proteins SLP65 and CIN85 is a key signaling transducing element for the BCR and the focus of this doctoral thesis.
2.3 SLP65: A central adaptor molecule for BCR signaling
Adaptor proteins is a term commonly used for proteins that possess no inherent enzymatic activity but contain protein-protein or protein-lipid interaction domains. These can influence biological processes by targeting ligands to specific subcellular localizations, regulate their enzymatic activities allosterically, or enable the formation of protein complexes (reviewed in [95]). Adaptor proteins determine the spatial and temporal organization of protein complexes in cells to a large extent (reviewed in [52]). Enzymes and adaptor proteins cannot be strictly separated because many enzymes possess also adaptor protein properties.
The adaptor SH2-domain containing leukocyte protein of 65 kDa (SLP65), also called B cell linker (BLNK), B cell adaptor containing SH2 domain (BASH) or B cell activation (bca) gene product, was identified as a protein heavily phosphorylated upon BCR stimulation [192, 47, 51, 49]. SLP65 is essential for both, B cell development and activation [47, 195, 78, 110]. It is phosphorylated by Syk, but not by the Tec-family kinase Btk [47]. For chicken SLP65, nine phosphorylated tyrosine residues can be detected, seven of which are conserved in human SLP65 [122].
While the lack of SLP65 leads always to severe impairment of B cell development and function, the degree of this impairment depends on the investigated species. In the chicken B cell line DT40, SLP65 deficiency abolishes completely Ca2+ mobilization upon BCR stimulation [75]. In contrast, there is still Ca2+ mobilization detectable in SLP65-deficient mice, even if it is reduced compared to wildtype mice [127, 78]. This remaining Ca2+signaling is attributed to the complementation of SLP65 by other adaptors expressed in mouse B cells, like SLP76 [170], or alternative pathways of Ca2+ mobilization, e.g. CD19 signaling ([38] and reviewed in [95]. As a result of an impaired B cell development, SLP65-deficient mice show reduced B cell numbers [127]. The immune response to T-cell-independent antigens is abolished [197]. Human patients with SLP65-deficiency show a severe immunodeficiency, they do not have mature B cells and suffer from agammaglobulinemia [110].
SLP65 mediates Ca2+ influx by assembly of the Ca2+ initiation complex, consisting of Bruton’s ty-
rosine kinase (Btk), phospholipase C-γ2 (PLC-γ2), and SLP65 itself [97].
Btk is a protein tyrosine kinase [142]. Mutations in the Btk gene lead to immunodeficiency in form of X-linked agammaglobulinemia (XLA) [185, 123]. On cellular level, impaired Btk activation results in reduction of BCR-induced Ca2+ signaling [141]. Btk has been shown to be activated by two mecha- nisms upon BCR stimulation. Firstly, Btk is recruited to the plasma membrane by its binding to the membrane lipid PIP3 which is produced after BCR stimulation [151]. Secondly, Btk is additionally ac- tivated by tyrosine phosphorylation in its activation loop, carried out by Src-family kinases like Lyn [143]. By its SH2 domain, Btk can bind to the phospho-tyrosine (pY) 96 of SLP65 [62, 171]. This association is crucial for Btk function [172]. On one hand, it can promote Btk activity by bringing Btk to the proximity of Lyn and Syk what might lead to phosphorylation of the Btk activation loop [7]. On the other hand, the binding to SLP65 facilitates the interaction of Btk with its substrate PLC-γ2 [62, 97].
PLC-γ2 is activated by phosphorylation by Syk [175] and Btk [46]. This phosphorylation processes are dependent on the presence of phosphorylated SLP65 that serves as a platform for the assembly of the PLC-γ2-Btk complex (Ca2+initiation complex) [75, 96]. The SH2 domains of PLC-γ2 bind to pY178 and pY189 of SLP65 [47, 76]; its C2 domain binds to pY119 [39]. It has been shown that SLP65 assembles Btk and PLC-γ2 in cis, as both proteins have to be bound to the same SLP65 molecule [24]. After full activation, PLC-γ2 cleaves the plasma membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) into the second messenger molecules inositol trisphosphate (IP3) and diacyl-glycerol (DAG) [69]. IP3 binds to the IP3 receptor in the membrane of the endoplasmic reticulum (ER), resulting in the release of Ca2+
from the ER lumen to the cytosol by the opening of ion channels (reviewed in [11]). The emptying of the ER Ca2+ reservoir leads in turn to the Stromal interaction molecule (STIM)-mediated opening of store-operated Ca2+channels (SOCs) in the plasma membrane. This results in the influx of extracellular Ca2+(reviewed in [105] and [155]).
The influx of Ca2+ and the generation of DAG initiate signaling cascades that lead in the end to the activation of a variety of transcription factors. The elevated intracellular Ca2+concentration is crucial for the activation and nuclear translocation of the transcription factors NfκB and NFAT [4]. Furthermore, high Ca2+levels cooperate with DAG binding to activate the protein kinase C-β (PKC-β) ([120]. PKC-β phosphorylates the protein CARD11, followed by the activation of the Inhibitor ofκB kinase (IKK) and the nuclear translocation of the transcription factor NfκB ([132], reviewed in [163]).
The DAG-mediated activation of PKC-β triggers as well signaling pathways leading to the activation of the mitogen-activated protein kinases (MAPKs) JNK and p38 [56, 61]. DAG provides also a docking site for RasGRP proteins [29]. This leads in turn to activation of the MAPK pathway via Ras, result- ing in the activation of different transcription factors by the extracellular signal-regulated kinase (ERK) [61]. ERK can alternatively be activated by an adaptor function of SLP65 independent of the Ca2+
SOS activates Ras and initiates MAPK activation [192, 47, 5]. The MAPKs activate many transcription factors that play a role in lymphocyte activation, proliferation, and differentiation (reviewed in [148] and [113]). Figure 2.2 gives an overview of the described signaling pathways initiated by BCR stimulation and mediated by SLP65.
Depending on the developmental stage of the B cell and other extracellular signals, BCR signaling can support activation and proliferation, but under certain circumstances also anergy or apoptosis (e.g. in autoreactive B cells) (reviewed in [118]).
As an upstream event for all of the described pathways, the phosphorylation of SLP65 is a key step for BCR signaling. While the signaling pathways downstream of SLP65 are quite well understood, there is still one unsolved problem regarding this molecule itself. It is unclear how SLP65 is translocated from the cytosol to the plasma membrane upon BCR stimulation. The adaptor protein homolog of SLP65 in T cells, SLP76, has been shown to be targeted to the plasma membrane by binding to the membrane protein LAT via the adaptor GADS [103]. However, for SLP65 no such membrane anchor could be identified yet.
The question of SLP65 membrane recruitment requires a closer look at the SLP65 domain structure and its multiple interactions.
2.4 The SLP65 regions and their functions
SLP65 consists of a positively charged N-terminal region, a central proline rich region and a C-terminal SH2 domain (Figure 2.3).
The N-terminal region of SLP65 has been described to form a leucine zipper which associates presumably with a membrane protein to provide SLP65 membrane anchoring [87]. In contrast, our analyses pointed to the direction that the N-terminus is not primarily a protein-protein-interaction-mediating leucine zipper but a lipid interaction module. It anchors SLP65 to membranes, dependent on their curvature [40, 136].
The N-terminus is crucially required for the translocation of SLP65 to the plasma membrane. It has been shown that the SLP65 N-terminus can be substituted by a lipid binding device, namely a myristoylation signal [87] or the membrane anchoring domain of TIRAP [67, 18].
The C-terminal SH2 domain of SLP65 is essential for the anchoring of SLP65 to the plasma membrane and the BCR [2]. It binds to the non-ITAM pY204 in the tail of Igα[42, 79]. However, in a chimeric CD8-Igα/CD8-Igβ model, the SLP65-Igαinteraction was partially dispensable, questioning its relevance for SLP65 membrane and BCR recruitment under all conditions [135]. Consistent with this observation, transgenic mice with a Y-to-F exchange of Y204 of Igαshowed that the SLP65-Igαinteraction is only important for T-cell-independent immune responses [129]. The SH2 domain does also bind to the kinase HPK1 [184, 154] and Syk. The Syk-SLP65 interaction can occur without Syk phosphorylation [2].
Fig. 2.2: Overview of the SLP65 functions in BCR signaling. The BCR is activated by binding to its antigen. This leads to phosphorylation of two tyrosines residues in the ITAM motif which display a binding site for the SH2 domain of the kinase Syk. Activated Syk phosphorylates the adaptor protein SLP65 which is in a steady complex with the adaptor CIN85. After its phosphorylation, SLP65 constitutes a platform for the assembly of the kinase Btk and the lipase PLC-γ2, resulting in PLC-γ2 activation.
PLC-γ2 hydrolyzes the membrane lipid PIP2 to produce the second messengers DAG and IP3. IP3
mediates the influx of Ca2+ from the ER to the cytosol. DAG initiates the activation of the kinase PKC-β, this process is enhanced by the elevated Ca2+concentration. PKC-βactivation and Ca2+ influx trigger signaling cascades that lead in the end to the activation of the transcription factors NFAT, NfκB and of the MAP kinases p38 and JNK. The activation of the MAP kinase Erk is triggered either by SOS, a molecules recruited by SLP65 via Grb2, or alternatively by DAG via the recruitment of RasGRP (not depicted). The activation of transcription factors and MAP kinases changes the transcriptional program of the B cell and may promote activation and proliferation, but also anergy, depending on other extracellular signals.
Fig. 2.3: Schematic domain structure of SLP65. SLP65 possesses a positively charged N-terminal region (aa 1-45, labeled by +). The following region of the protein is proline rich (aa 46-345, not labeled). This region contains two PXXXPR motifs as binding sites for CIN85 (aa 242-247 and 307-312, depited by black ovals). The C-terminus of the protein forms a SH2 domain (aa 346-453, SH2). The upper image displays the known secondary structure elements in SLP65. Y represents a tyrosine residue that is phosphorylated upon BCR stimulation. The pentagons represent proline residues of the two CIN85 binding sites.
The central proline rich region (PRR) of SLP65 contains the tyrosine residues that are phosphorylated upon BCR stimulation and several proline-containing interaction motifs [121]. Three motifs with the consensus sequence PXXXPR have been shown to interact with SH3 domains of the adaptor protein CIN85 [122]. The second and third of these three motifs are responsible for the interaction of SLP65 with CIN85, while the first motif is dispensable [122]. The SLP65-CIN85 interaction is required for ef- ficient SLP65 plasma membrane recruitment and phosphorylation and hence for the induction of Ca2+
mobilization [121]. So the SLP65-CIN85 interaction constitutes a third functionality that is important for SLP65 plasma membrane recruitment, besides SLP65 N-terminus and SH2 domain.
2.5 CIN85: a multi-domain adaptor protein with still mysterious functions
The human Cbl-interacting protein of 85 kDa (CIN85) has been cloned and characterized by Take, Watan- abe and colleagues. [176, 189]. Simultaneously, its homolog in rats was cloned by Gout and colleagues and B¨ogler and colleagues who termed the protein Regulator of ubiquitous kinase (Ruk) respectively SH3 domain containing gene expressed in tumorigenic astrocytes (SETA) [55, 15]. From the beginning on, CIN85 was characterized as an adaptor protein binding to the ubiquitin E3 ligase c-Cbl [176]. Together with its homolog CD2AP, it forms a family of adaptor proteins [176, 55]. Its domain structure was elucidated very soon after the cloning of the gene on the basis of bioinformatics [176, 189]. CIN85 has three N-terminal SH3 domains followed by a proline-rich region (PRR) and a C-terminal coiled-coil (CC) domain (Figure 2.4). The structure prediction could be confirmed by NMR spectroscopy studies. The structure of all three SH3 domains is known ([1, 133]. Structure and oligomerization state of the CC were solved in a collaboration of C. Griesinger’s and our group (see 4.10).
Fig. 2.4: Schematic domain structure of CIN85. CIN85 possesses three N-terminal SH3 domains (aa 1- 328, SH3), followed by a proline-rich region (aa 329-599, PRR). The C-terminal part of CIN85 consists of a coiled-coil domain (aa 600-665, CC). The upper image displays the known secondary structure elements in CIN85.The pentagons represent proline residues of the intramolecular SH3 domain binding site within the PRR.
CIN85 is an ubiquitously expressed protein that occurs in several isoforms [55]. The largest isoform contains all the domains described above, while the other isoforms lack one, two, or all three SH3 do- mains ([19, 44, 108]. The SH3 domains of CIN85 bind to atypical proline motifs containing the consensus sequence PXXXPR (X= any aa) [92, 94]. They have also been shown to bind to ubiquitin ([13, 168].
The proline-rich region of CNI85 has been implicated to have an autoregulatory function. It can bind to the first and second SH3 domain of CIN85 ([92, 17]. This intramolecular interaction can compete with the SH3-domain-PRR-mediated interaction of CIN85 with PI3K p85-α[17]. The CC can mediate homo-oligomerization of CIN85 as well as association with the CIN85-homolog CD2AP [189, 16, 48]. The CC has also been described to mediate membrane association by phosphatidic acid (PA) binding [200].
CIN85 has been reported to interact with various proteins (reviewed in [64]). As adaptor protein, CIN85 is thought to exert its functions by bringing its interaction partners in spatial proximity. Mass spectro- metric screens show that the CIN85 SH3 domains are redundant in their specificity [65, 20], at least under in vitro conditions. Corresponding to this finding, it was shown biochemically that one CIN85 protein can cluster several c-Cbl molecules [92]. While the knowledge of CIN85 structure and of its interaction partners increased, the exact cellular functions of CIN85 were less clear and sometimes controversial.
The binding to SLP65 and c-Cbl was reported already in 2000 [189], and it was speculated that these interactions might influence PLC-γ2 activity in B cells. Nevertheless, the focus of CIN85 function was on its role for Epidermal growth factor receptor (EGFR) signaling for the next decade [166, 92, 91]. Upon the c-Cbl-mediated recruitment of CIN85 to the activated EGFR, CIN85 is described to cluster c-Cbl and endophilin molecules via interaction of their respective proline motifs with the CIN85 SH3 domains [166]. By this mechanism, CIN85 recruitment has been shown to promotes receptor internalization of the activated EGFR and other receptor tyrosine kinases [166, 131, 174]. However, the importance of CIN85 for EGFR endocytosis was negated by other reports ([63, 70, 77] and is regarded controversially (reviewed in [64]). Besides the endocytotic processes itself, the intracellular trafficking of the endocytosed EGFR has been reported to depend on SH3-mediated interactions of CIN85 with various interaction partners
ficking [160].
The subcellular localization of CIN85 is still under dispute. CIN85 has been observed to be present in endosomes, COPI-coated vesicles or peripheral structures as lamellipodia and invadopodia, depending on the cell type and the CIN85 expression level [200, 63, 157, 117]. It is possible that the subcellular localization of CIN85 is determined by its respective binding partners in the investigated model systems.
The functions attributed to CIN85 depend mainly on its ligands. Because many of them are known to participate either in endocytosis or in Golgi-apparatus-linked vesicular transport, it is likely that CIN85 has an influence on these processes [64]. There have been some studies on the CIN85 function in immune cells. CIN85 has been shown to interact with the cytoplasmic tail of the receptor CD2 [16]. In T cells, CIN85 can link CD2 to the cytoskeleton by binding to the actin capping protein CapZ. This influences potentially the TCR-induced cytoskeleton reorganization [71]. CIN85 has also been implicated to sta- bilize CD2 expression at the plasma membrane [179]. In neutrophils, CIN85 has been reported to be recruited to the activated FcγRIIa and to promote internalization and degradation of this receptor in a c-Cbl dependent manner [107].
CIN85 has also been implicated in the signaling of the FcRI in mast cells, a receptor that belongs to the Multichain immune recognition receptor (MIRR) family, as BCR and TCR [134]. It was shown that CIN85 contributes to endocytosis and intracellular trafficking of the FcRI. Overexpression of CIN85 accelerated the FcRI internalization, impairing the FcRI-mediated degranulation of mast cells [112].
Furthermore, CIN85-overexpression reduced the level of Syk in mast cells, thereby diminishing also PLC- γ phosphorylation and Ca2+influx upon FcRI stimulation. [130]. In contrast to the inhibitory effect of CIN85 on the FcRI, the knockdown of CIN85 in the chicken B cell line DT40 led to an impaired BCR signaling, especially if it was done in cells deficient for the CIN85 homolog CD2AP [121]. This suggests that CIN85 might be a positive regulator in BCR signaling. No effect of CIN85 on BCR endocytosis was found [119, 18]. It has further been demonstrated that CIN85 interacts with the phosphatase SHIP1 in B cells, leading to the hypothesis that CIN85 might downregulate BCR signaling by recruiting this negative regulator to the activated BCR [20].
So the overall image of existing literature suggested that CIN85 might be a negative as well as a positive regulator of BCR signaling. Nevertheless, it was clear that the interaction of CIN85 with SLP65 had an enhancing effect on the BCR signaling cascade [121]. The aim of my doctoral thesis was to elucidate the mechanism by which CIN85 supports SLP65, gaining thereby general insight into the CIN85 function.
2.6 Aim of this study
Plasma membrane recruitment and phosphorylation of the adaptor protein SLP65 are crucial key steps of the BCR signaling and hence efficient B cell activation. These processes have been shown to depend on the interaction of SLP65 with the other adaptor protein CIN85. Nevertheless, it was totally unclear how the interaction with CIN85 contributes to SLP65 function. The aim of my doctoral thesis was the elucidation of the molecular mechanism by which CIN85 supports the function of SLP65 in BCR signaling.
To get inside into CIN85 function in B cells, I dealt with the following specific questions:
1. Does CIN85 possess protein or lipid ligands to target SLP65 to specific subcellular compartments?
The CIN85 protein interactome was investigated by mass spectrometric analysis. Membrane association was assessed by subcellular fractionation experiments and lipid binding studies.
2. Which domains of CIN85 are essential for its support of SLP65? Can CIN85 binding be substituted by equipping SLP65 directly with additional functional domains? These points were addressed genetically by the generation of chimeric proteins and subsequent analysis of their functionality in the BCR pathway.
3. Can the structure of CIN85 explain its function? The structure of the C-terminal CIN85 coiled- coil domain was elucidated in cooperation with Prof. C. Griesinger’s group . With this knowledge, the influence of single amino acids for CIN85 function could be examined.
3.1 Material
3.1.1 Instruments
Tab. 3.1: Instruments used in this study
Name Manufacturer
Agarose Gelelectrophoresis System Peqlab
Balance BP61 Sartorius
Balance H95 Sartorius
Balch homogenizer MPI BPC, G¨ottingen
BioPhotometer Eppendorf
Cell culture incubator HeraCell 150 CO2 Heraeus
Cell disruption vessel 45 ml Parr Instrument
Chemi Lux Gel Imager Intas Science Imaging
Confocal laser scanning microscope TCS SP2 Leica Microsystems
Countess cell counter Invitrogen
Electrophoresis power supply EPS 301 Amersham BioSciences Electrophoresis System Hoefer SE600 Amersham
Flow cytometer LSR II Becton Dickinson
Freezer Platilab 340 Angelantoni Industrie
GenePulser II electroporation system Bio-Rad Laboratories
Ice machine Ziegra
Incubation shaker Unitron Infors
Incubator Kelvitron t Heraeus
Laminar flow cabinet HERA safe Heraeus
Light microscope TELAVAL 31 Zeiss
LSR II BD Biosciences
Magnetic stirrer M21/1 Framo Ger¨atetechnik
Mastercycler epgradient Eppendorf
Microcentrifuge 5415D Eppendorf
Microcentrifuge 5417R Eppendorf
Microplate reader PowerWave 340 BioTek
Mini PROTEAN Tetra Cell Bio-Rad
Multifuge 3SR Heraeus
NanoDrop 2000 Thermo Scientific
Name Manufacturer
pH-Meter inoLab WTW
Pipettes Eppendorf
Rocking shaker Neolab
Rotor SW40 Ti Beckman
Rotor SW41 Ti Beckman
Semiphor Transphor Unit TE77 Amersham Bioscience
Shaker 3006 GFL
Sonicator Bandelin Sonoplus
Sorvall RC 3B Plus Sorvall
Thermomixer comfort Eppendorf
Ultracentrifuge Optima L-70k Beckman
Ultracentrifuge Optima LE 80k Beckman
UV-illuminator Intas systems
Vortex Genie 2 Scientific industries
Water bath GFL
Water Purification System Milli-Q Millipore, Sartorius
3.1.2 Software
Tab. 3.2: Software used in this study
Name Manufacturer Application
Chemostar professional Intas Immunoblot documentation
Coreldraw Corel Corporation Graphic editing
FACSDiva BD Biosciences Flow cytometry recording
Flowjo Treestar Flow cytometry analysis
ImageJ W. Rasband Image processing
Leica confocal software Leica Microscopy image processing
LaTeX L. Lamport Text processing
Microsoft office2010 Microsoft Text processing
pDraw32 Acaclone software DNA sequence information processing
Tab. 3.3: Consumables used in this study
Name Manufacturer
Blotting Paper Whatman GE Healthcare
CELLSTAR 96 well suspension culture plates Greiner bio-one
CELLSTAR dishes 60 mm, 100 mm, 145 mm Greiner bio-one
CELLSTAR serological pipettes 2 ml, 5 ml, 10 ml, 25 ml Greiner bio-one
CELLSTAR tubes, 15 ml, 50 ml Greiner bio-one
Cyro.S tubes Greiner bio-one
Dialysis tubing SERVAPOR Serva
Dishes for bacteria, 92 mM Sarstedt
Filter tips Greiner bio-one
Microscope imaging chambers, 4 wells Lab Tek
Nitrocellulose membrane Hybond ECL Amersham Biosciences
Parafilm American National Can
PCR tubes, 0.2 ml Sarstedt
Photometer cuvettes Roth
Pierce centrifuge columns Thermo scientific
Pipette tips Greiner bio-one
Reaction tubes, 1.5 ml, 2 ml Greiner bio-one
Scalpel B. Braun
Sterile filter Filtropur, S 0.2 and S 0.45 Sarstedt
Syringe Omnifix ,1 ml B. Braun
Syringes, 5 ml, 10 ml BD Biosciences
Tubes for flow cytometry, 5 ml Sarstedt
Tubes, 14 ml Greiner bio-one
Ultracentrifugation tubes 9/16 x 3 3/4” Beckman
Vivaspin 500 Sartorius
3.1.4 Chemicals and reagents
Tab. 3.4: Chemicals and reagents used in this study
Name Manufacturer
Acetic acid (HOAc) Roth
Acrylamide/bis-Acrylamide Rotiphorese Gel 30 Roth
Agar-Agar Roth
Agarose Peqlab
Ammonium persulfate (APS) Roth
Ampcillin Roth
L-Arginine:HCl (13C6) Cambridge Isotope Lab.
Bleomycin (Bleocin) Merck
Bovine serum albumin (BSA) Serva
Brom-chlor-indoxyl-β-D-galactosid (X-gal) Roth
Bromophenol blue Merck
Bovine serum albumin (BSA) solution NEB
Calcium chloride (CaCl2) Merck
Chicken serum Sigma
Coomassie Brilliant Blue R-250 Roth
Dimethyl sulfoxide (DMSO) Roth
DNA Ladder GeneRuler 1kb Fermentas
Deoxynucleoside triphosphate (dNTP) mix NEB
Dithiothreitol (DTT) Roth
n-Dodecylβ-D-maltoside Sigma-Aldrich
Ethylenediaminetetraacetic acid (EDTA) Roth
Ethanol Roth
Ethidium Bromide Roth
Fetal Calf Serum (FCS), dialyzed PAN Biotech
Fetal Calf Serum (FCS) Biochrom
D-Glucose Roth
Glycerol Roth
Glycine Roth
Hydrogen peroxide (H2O2) Roth
Hydrochloric acid (HCl) Roth
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) Roth
Hexadimethrine bromide (polybrene) Sigma-Aldrich
Igepal CA-630 (NP40) Sigma-Aldrich
Immersion oil Merck
INDO-1 AM Life technologies
Invisorb Spin Plasmid Mini Two Invitek
Isopropyl-β-D-thiogalactopyranoside (IPTG) Roth
Isopropanol Roth
Kanamycin Roth
Luminol Sigma-Aldrich
L-Lysine:2HCl (4,4,5,5-D4) Cambridge Isotope Lab.
µMACS GFP Tagged Protein Isolation kit Miltenyi
Magnesium acetate (Mg(OAc)2) Roth
Magnesium chloride (MgCl2) Roth
Methanol (MeOH) Roth
NEBuffers 1,2,3,4,cut smart NEB
NuPAGE LDS sample buffer Invitrogen
NuPAGE sample reducing reagent Invitrogen
Phosphatidic acid (PA) coated beads Echelon
p-Coumaric acid Sigma-Aldrich
Penicillin Sigma-Aldrich
Phusion HF reaction buffer NEB
Pluronic F-127 Life technologies
Potassium chloride (KCl) Roth
Potassium dihydrogen phosphate (KH2PO4) Merck
Prestained Protein Marker Broad Range NEB
Protease Inhibitor Cocktail P2714 Sigma-Aldrich
Protein A/G agarose beads Santa Cruz
Protino Ni-IDA 1000 Macherey-Nagel
Pure Yield Plasmid Midiprep System Promega
Puromycin InvivoGEN
Sodium acetate (NaOAc) Roth
Sodium azide (NaN3) Roth
Sodium chloride (NaCl) Roth
Sodium dodecyl sulfate (SDS) Roth
Sodium fluoride (NaF) Roth
di-Sodium hydrogen phosphate (Na2HPO4) Roth
Name Manufacturer
Sodium hydroxide (NaOH) Roth
Sodium orthovanadate (Na3VO4) Sigma-Aldrich
Strep-Tactin Superflow high capacity IBA
Strep-tag elution buffer IBA
Streptomycin Sigma-Aldrich
Sucrose Roth
T4 DNA Ligase buffer NEB
Tetramethylethylenediamine (TEMED) Roth
TOPO TA Cloning Kit Invitrogen
Trans-IT Mirus
Tris(hydroxymethyl)-aminomethane (Tris) Roth
Triton X-100 Roth
Trypsin Gibco
Trypton/Pepton Roth
Tween 20 Roth
Wizard SV Gel and PCR clean up Kit Promega
Yeast extract Roth
3.1.5 Buffers and solutions
Tab. 3.5: Buffers and solutions used in this study. All %-indications refer to weight per volume.
Name Composition
Antibody dilution solution 1% BSA, 0.01% NaNa3 in TBS-T
Blotting buffer 48 mM Tris, 39 mM glycine, 0.0375% SDS, 0.001% NaN3, 20% MeOH
Cell lysis buffer 50 mM Tris, 50 mM NaCl, 5 mM NaF, 1 mM Na3VO4, 1:50 Protease Inhibitor, 0.5% NP40, pH 8.0
Cell lysis buffer for strep-tag AP Cell lysis buffer + 0.5% n-dodecylβ-D-maltoside Coomassie staining solution 40% MeOH, 10% HOAc, 0.1% Coomassie Brilliant Blue
R-250
DNA loading buffer (6x) 60 mM EDTA, 10 mM Tris/HCl, 60% glycerol, 0.03%
Bromophenol blue, pH 7.6
ECL solution composition 4 ml ECL solution A, 400µl ECL solution B, 1.2µl H2O2
(30%)
ECL solution SA 100 mM Tris/HCl, 0.28 mM Luminol, pH 8.6
ECL solution SB 6.7 mM p-coumaric acid in 100% DMSO
Homogenization buffer 250 mM Sucrose, 20 mM KCl, 25 mM HEPES, 2.5 mM Mg(OAc)2, 2.5 mM DTT, 1:50 Protease Inhibitor, pH 7.4 Krebs-Ringer solution with
CaCl2
140 mM NaCl, 10 mM D-Glucose, 10 mM HEPES, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, pH 7.4
Laemmli loading buffer (5x) 500 mM DTT, 150 mM Tris/HCl, 50% Glycerol, 15% SDS, 0.05% Bromophenol blue, pH 6.8
Lew buffer 300 mM NaCl, 50 mM NaH2PO4, pH 8.0
PBS 137 mM NaCl, 2.4 mM KCl, 4.3 mM Na2HPO4, 1.4 mM
KH2PO4, pH 7.4
Resolving gel solution 375 mM Tris/HCl, 10% Acrylamide/bis-Acrylamide, 0.1%
APS, 0.1% TEMED, pH 8.8
SDS-PAGE running buffer 192 mM glycine, 25 mM Tris, 0.1% SDS
Stacking gel solution 125 mM Tris/HCl, 4.8% Acrylamide/bis-Acrylamide, 0.1%
APS, 0.1% TEMED, pH 6.8
TAE buffer 40 mM Tris/HOAc, 10 mM NaOAc, 1 mM EDTA, pH 7.8
TBS-T 137 mM NaCl, 20 mM Tris, 0.1% Tween20, pH 7.6
Western blot blocking solution 5% BSA, 0.01% NaN3in TBS-T
3.1.6 Antibodies
Tab. 3.6: Antibodies used in this study
Name Manufacturer
α-Akt, rabbit monoclonal (C73H10) Cell signaling technologies α-Blnk, mouse monoclonal (2B11) BD Biosciences
α-chicken IgM, mouse (M4) Bethyl Laboratories α-CIN85, rabbit polyclonal (C6115) Sigma-Aldrich α-Clathrin heavy chain, mouse monoclonal (23/Clathrin) BD Biosciences α-GFP, mouse monoclonal (7.1+13.1) Roche
α-GM130, mouse monoclonal (35/GM130) BD Biosciences α-Grb2, mouse monoclonal (81/GRB2) BD Biosciences
α-HA, rat monoclonal (3F10) Roche
α-human IgM, F(ab’)2Fragment, mouse Jackson Immune Research Lab.
α-LAMP1, mouse monoclonal (H4A3) BD Biosciences α-Lyn, mouse monoclonal (42/Lyn) BD Biosciences
Name Manufacturer α-Rab5, mouse monoclonal (621.1) Synaptic Systems α-α-Tubulin, mouse monoclonal (B512) Sigma-Aldrich α-VAMP7, rabbit polyclonal (LS-B88839) LifeSpan BioSciences α-mouse IgG (HRP-conjugated), goat Southern biotech α-rabbit IgG (HRP-conjugated), goat Southern biotech
3.1.7 Enzymes
Tab. 3.7: Enzymes used in this study
Name Manufacturer
AgeI HF NEB
Alkaline phosphatase, calf intestinal (CIP) NEB
BamHI HF NEB
BglII NEB
ClaI NEB
DpnI NEB
HindIII HF NEB
NdeI NEB
NotI HF NEB
Phusion High-Fidelity DNA Polymerase NEB
PvuI NEB
T4 DNA ligase NEB
Taq PCR Master Mix Qiagen
XhoI NEB
3.1.8 Media
Tab. 3.8: Media used in this study
Name Manufacturer/Composition
RPMI-1640 Merck
DMEM Merck
SILAC RPMI-1640 Medium Thermo Scientific
Lysogeny broth (LB medium) 10 g/L trypton/Pepton, 5 g/L yeast extract, 10 g/L NaCl
LB-Agar 15 g Agar-Agar in 1L LB medium
Tab. 3.9: Oligonucleotides used as PCR primers in this study. All oligonucleotides were manu- factured by MWG-Biotech.
Name Sequence
BglII SLP65 fw TAATAGATCT CGACAAGCTT AATAAAATAA
CCGTCC
CD2AP CC fw Bam TTCAGGATCC CATGGAAATC AAAGCTAAAG
TGGA
CD2AP CC Nde fw TTCACATATG ATGGAAATCA AAGCTAAAG
CD2AP rev Bam ME frame AAAAAAGCTG TCCTGTCTTC GGATCCACTT
Cin CC 631 Not rev TATGCGGCCG CTCATTTCTG CTGGTCCTTC
ATGGTCT
Cin CC 632 Bgl fw TTCAAGATCT CATGCGAGAG ATTAAACAGT
TATTGTCT
Cin CC Bam 631 rev CGAGGGATCC TTCTGCTGGT CCTTCATGGT CT
Cin CC Bgl fw TTCAAGATCT CATGGAAGGA AAACCAAAGA
TGGAGCCT
Cin PR/CC Not rev with stop ATGCGGCCGC TCATTTTGAT TGTAGAGCTT TCTT
CIN85 CC fw Nde TTCGCATATG GAAGGAAAAC CAAAGATGGA
GCCT
CIN85 CC fwd Bam AAAGGATCCA CCATGGAAGG AAAACCAAAG
ATGGAGCCT
CIN85 CC rev Xho with stop TTCGCTCGAG TTATTTGATT GTAGAGCTTT CTT
Cin85 PR/CC Bam fw TTCGTGGATC CCATGGACTT TGAAAAGGAA GGG
Cin85 PR/CC Rev CGAGGGATCC TTTGATTGTA GAGCTTTCTT
Cin85 SH3 rev CGAGGGATCC GGTGGAAGTA ACTTCACGAA
Cin85SH3 ATG Blg fw TTCGTAGATC TCATGGTGGA GGCCATAGTG GA
CinCC L619A fw ACACAGGTCC GCGAGGCGAG GAGCATCATC
GAGAC
CinCC L619A rev GTCTCGATGA TGCTCCTCGC CTCGCGGACC
TGTGT
CinCC L619K fw ACACAGGTCC GCGAGAAGAG GAGCATCATC
GAGAC
Name Sequence
CinCC L619K rev GTCTCGATGA TGCTCCTCTT CTCGCGGACC
TGTGT
CinPR Bam rev CGAGGGATCC ATCTTTGGTT TTCCTTCCGT
HAtag Cin CC Bgl fw TTCAAGATCT CATGTACCCA TACGACGTCC
CAGACTACGC TGAAGGAAAA CCAAAGATGG AGCCT
hSlp rev stop Xho TTCTCTCGAG TTATGAAACT TTAACTGCAT
hSLP65woStopAge ATATACCGGT GGATCTGAAA CTTTAACTGC
ATACTTCAG
M13fw GTAAAACGAC GGCCAG
M13rev CAGGAAACAG CTATGAC
NotI SLP65 rev TAATGCGGCC GCTTATGAAA CTTTAACTGC
ATACTTCAG
Overlap slpCinCC 632 fw AAGTATGCAG TTAAAGTTTC ACGAGAGATT AAACAGTTAT TGTCT
Overlap slpCinCC632 rev AGACAATAAC TGTTTAATCT CTCGTGAAAC TTTAACTGCA TACTT
pEGFPN1 for GTCGTAACAA CTCCGCCC
pEGFPN1 rev GTCCAGCTCG ACCAGGATG
pMSCVfw CCCTTGAACC TCCTCGTTCG ACC
pMSCVrev CAGACGTGCT ACTTCCATTT GTC
Raf1 PA Bam fw TTCAGGATCC CATGAGGAAT GAGGTGGCTG
TTCT
Raf1 PA Bam rev TGAAGGATCC CCTGATCCCT CGCACCACTG
GGTCAC
SCOC CC Bam fw TTCAGGATCC CATGATGAAT GCCGACATGG AT
SCOC CC Bam rev TGAAGGATCC TTACGTTTGG ATTTGGTATC G
SCOC E93V K97L fwd GCCGAAAATC AGGTGGTACT GGAGGAATTA
ACCCGTCTGA TCAAC
SCOC E93V K97L rev GTTGATCAGA CGGGTTAATT CCTCCAGTAC
CACCTGATTT TCGGC
SCOC N125L N132V fw TGCCGTAAAA GAGGAGCTTC TGAAACTGAA
AAGTGAGGTT CAAGTGCTGG GC
SCOC N125L N132V rev GCCCAGCACT TGAACCTCAC TTTTCAGTTT
CAGAAGCTCC TCTTTTACGG CA
Slp dN (46) Bam ATG fw TTCGGGATCC CATGAGTGTT CCTCGAAGGG ACTACG
Slp dN (46) Bgl ATG fw TTCGAGATCT CATGAGTGTT CCTCGAAGGG
ACTACG
Slp EcoRI rev AAGTGAATTC TTATGAAACT TTAACTGCAT A
SLP65CIN85CC OL fw AAGTATGCAG TTAAAGTTTC AGAAGGAAAA
CCAAAGATGG AGCCT
SLP65CIN85CC OL rev AGGCTCCATC TTTGGTTTTC CTTCTGAAAC
TTTAACTGCA TACTT
SLP65CIN85PRR OL fw AAGTATGCAG TTAAAGTTTC AGACTTTGAA
AAGGAAGGGA AT
SLP65CIN85PRR OL rev ATTCCCTTCC TTTTCAAAGT CTGAAACTTT
AACTGCATAC TT
Slp76 Age rev TGAAACCGGT GGGTACCCTG CAGCATGCGT TAA
Slp76 Bam fw TTCAGGATCC CGCACTGAGG AATGTGCCC
slpdN Bam fw frVB TTCAGGATCC AGTGTTCCTC GAAGGGACTA CG
Slprev fl Xho mit Stop TTCGCTCGAG TTATGAAACT TTAACTGCAT A
T7 TAATACGACT CACTATAGG
3.1.10 Plasmids
Tab. 3.10: Basic plasmids used in this study
Name Source,description Application
pCit-N1 Dr. M. Engelke, based on
pEGFP-N1 (BD Biosciences Clontech), eGFP exchanged for Citrine
Cloning, addition of Citrine tag to chimeric proteins
pET 16b TEV Dr. S. Becker Expression of recombinant
His-tagged proteins
pCR2.1 Invitrogen TA cloning
pABESpuro N-One-Strep Dr. V. Bremes Expression of electroporated cDNA in eukaryotic cells, includes N-terminal strep-tag
Name Source,description Application
pMSCV puro BD Biosciences Clontech Expression of retrovirally transduced cDNA in eukaryotic cells pMSCV puro plus
NotI-cutting site
Dr. M. Engelke, based on pMSCV puro, Not cleavage site added
Expression of retrovirally transduced cDNA in eukaryotic cells pMSCV bleo plus
NotI-cutting site
This thesis, puromycin resistance cassette exchanged for bleomycin resistance cassette (with ClaI and HindIII
restriction sites)
Expression of retrovirally transduced cDNA in eukaryotic cells
pMSCV puro NCit Dr. M. Engelke, based on pMSCV puro, Citrine added,
Expression of retrovirally transduced cDNA in eukaryotic cells, includes N-terminal Citrine tag
Tab. 3.11: Plasmid constructs used in this study. All proteins originated from human, with exception of VAMP7 (chicken). The plasmid were prepared for this thesis, if not indicated otherwise. The numbers indicate the amino acid position in the protein. General abbreviations:
SLP65-∆N means SLP65 aa 46-456. SLP65-M2,3 means SLP65 with amino acid exchanges R247A and R312A.
Name Description Source
pCitN1 SLP65-∆N SLP65-∆N, C-terminal Citrine tag
pCitN1 SLP65-∆N-M2,3 SLP65-∆N-M2,3, C-terminal Citrine tag
pCitN1 SLP65-M2,3 SLP65-M2,3, C-terminal Citrine tag
pCitN1 SLP65-wt SLP65-wt, C-terminal Citrine tag
pCitN1 SLP65-∆N RL SLP65-∆N R372L, C-terminal Citrine
Dr. M. Engelke
pCitN1 SLP76 SLP76, C-terminal Citrine pABES puro N-OneStrep
CD2AP
N-terminal Strep tag, CD2AP Dr. V. Bremes
pABES puro N-OneStrep CIN85
N-terminal Strep tag, CIN85 Dr. V. Bremes
pABES puro N-OneStrep CIN85 CC SLP65-∆N
N-terminal Strep tag, CIN85 594-665, SLP65-∆N
pCR2.1 CD2AP CC CD2AP 568-639
pCR2.1 CIN85 CC L619A SLP65-∆N-M2,3
CIN85 594-665 L619A, SLP65-∆N-M2,3 pCR2.1 CIN85 CC L619K
SLP65-∆N-M2,3
CIN85 594-665 L619K, SLP65-∆N-M2,3 pCR2.1 CIN85 CC
SLP65-∆N-M2,3
CIN85 594-665, SLP65-∆N-M2,3 pCR2.1 CIN85 PRR CIN85 329-599 pCR2.1 CIN85 PRR/CC CIN85 329-665
pCR2.1 CIN85 SH3 CIN85 1-328
pCR2.1 Raf PA Raf1 391-426
pCR2.1 SCOC CC SCOC 78-159
pCR2.1 SCOC CC Tetramer SCOC 78-159 N125L/N132V pCR2.1 SCOC CC Trimer SCOC 78-159 E93V/K97L pET16b CD2AP CC
SLP65-∆N
N-terminal His tag, CD2AP 568-639, SLP65-∆N
pET16b CIN85 CC N-terminal His tag, CIN85 594-665
pET16b CIN85 CC L619K N-terminal His tag, CIN85 594-665 L619K
pET16b CIN85 CC L619K SLP65-∆N
N-terminal His tag, CIN85 594-665 L619K, SLP65-∆N pET16b CIN85 CC
SLP65-∆N
N-terminal His tag, CIN85 594-665, SLP65-∆N
pET16b SLP65-∆N N-terminal His tag, SLP65-∆N S. Pirkuliyeva pET16b SLP65-wt N-terminal His tag, SLP65-wt S. Pirkuliyeva pET28a SCOC CC Strep
Tag
SCOC 78-159, C-terminal Strep tag
Dr. K. K¨uhnel
pHCMV-VSV-G coding for VSV glycoprotein Dr. V. Bremes pMSCV bleo Cerulean
VAMP7
N-terminal Cerulean, chicken VAMP7
Dr. M. Engelke
Name Description Source pMSCV bleo HA CIN85 CC
L619K SLP65-∆N-M2,3
N-terminal HA tag, CIN85 594-665 L619K,
SLP65-∆N-M2,3 pMSCV bleo HA CIN85 CC
SLP65-∆N-M2,3
N-terminal HA tag, CIN85 594-665, SLP65-∆N-M2,3 pMSCV bleo NCit
SLP65-∆N-M2,3 CIN85 CC 594-631
N-terminal Citrine, SLP65-∆N-M2,3, CIN85 594-631
pMSCV bleo VC Venus 156-239 Dr. M. Engelke
pMSCV bleo VC SLP65-∆N Venus 156-239, SLP65-∆N pMSCV bleo VC
SLP65-∆N-M2,3
Venus 156-239, SLP65-∆N-M2,3
pMSCV bleo VC SLP65-∆N-RL
Venus 156-239, SLP65-∆N R372L
pMSCV puro CD2AP CC SLP65-∆N CCit
CD2AP 568-639, SLP65-∆N, C-terminal Citrine
pMSCV puro CD2AP CC SLP65-∆N-M2,3 CCit
CD2AP 568-639,
SLP65-∆N-M2,3, C-terminal Citrine
pMSCV puro CD2AP CC CD2AP 568-639 pMSCV puro CIN85 CC
594-631 SLP65-∆N-M2,3 CCit
CIN85 594-631,
SLP65-∆N-M2,3, C-terminal Citrine
pMSCV puro CIN85 CC 594-631
CIN85 594-631
pMSCV puro CIN85 CC 632-665 SLP65-∆N-M2,3 CCit
CIN85 632-665,
SLP65-∆N-M2,3, C-terminal Citrine
pMSCV puro CIN85 CC 632-665
CIN85 632-665
pMSCV puro CIN85 CC SLP65-∆N-RL CCit
CIN85 594-665, SLP65-∆N R372L, C-terminal Citrine pMSCV puro CIN85 PRR
SLP65-∆N CCit
CIN85 329-599, SLP65-∆N, C-terminal Citrine
pMSCV puro CIN85 PRR SLP65-∆N-M2,3 CCit
CIN85 329-599,
SLP65-∆N-M2,3, C-terminal Citrine
pMSCV puro CIN85 PRR CIN85 329-599 pMSCV puro CIN85
PRR/CC SLP65-∆N CCit
CIN85 329-665, SLP65-∆N, C-terminal Citrine
pMSCV puro CIN85 PRR/CC SLP65-∆N-M2,3 CCit
CIN85 329-665,
SLP65-∆N-M2,3, C-terminal Citrine
pMSCV puro CIN85 PRR/CC
CIN85 329-665
pMSCV puro CIN85 SH3 SLP65-∆N CCit
CIN85 1-328, SLP65-∆N, C-terminal Citrine pMSCV puro CIN85 SH3
SLP65-∆N-M2,3 CCit
CIN85 1-328, SLP65-∆N-M2,3, C-terminal Citrine
pMSCV puro CIN85 SH3 CIN85 1-328 pMSCV puro CIN85CC
L619K SLP76
CIN85 594-665 L619K, SLP76, C-terminal Citrine
pMSCV puro CIN85CC SLP76
CIN85 594-665, SLP76, C-terminal Citrine pMSCV puro NCit CIN85
CC 594-631 SLP65-∆N-M2,3 CIN85 CC 632-665
N-terminal Citrine, CIN85 594-631, SLP65-∆N-M2,3 CIN85 632-665
pMSCV puro NCit CIN85 CC 632-665 SLP65-∆N-M2,3 CIN85 CC 594-631
N-terminal Citrine, CIN85 632-665, SLP65-∆N-M2,3 CIN85 594-631
pMSCV puro NCit CIN85 CC L619K SLP65-∆N-M2,3
N-terminal Citrine, CIN85 594-665 L619K,
SLP65-∆N-M2,3 pMSCV puro NCit CIN85
CC SLP65-∆N-M2,3
N-terminal Citrine, CIN85 594-665, SLP65-∆N-M2,3 pMSCV puro NCit
SLP65-∆N CIN85 PRR/CC
N-terminal Citrine, SLP65-∆N, CIN85 329-665
Name Description Source pMSCV puro NCit
SLP65-∆N-M2,3 CIN85 CC 594-631
N-terminal Citrine,
SLP65-∆N-M2,3 CIN85 594-631
pMSCV puro NCit
SLP65-∆N-M2,3 CIN85 CC
N-terminal Citrine, SLP65-∆N-M2,3, CIN85 594-665
pMSCV puro NCit SLP65-∆N-M2,3 CIN85 PRR/CC
N-terminal Citrine, SLP65-∆N-M2,3, CIN85 329-665
pMSCV puro NCitrin SLP65-∆N
N-terminal Citrine tag, SLP65-∆N
Dr. V. Bremes
pMSCV puro NCitrin SLP65-wt
N-terminal Citrine tag, SLP65-wt
Dr. V. Bremes
pMSCV puro PH SLP65-∆N CCit
PLC-δ PH domain, SLP65-∆N, C-terminal Citrine tag
pMSCV puro PH SLP65-∆N-M2,3 CCit
PLC-δ PH domain,
SLP65-∆N-M2,3, C-terminal Citrine tag
pMSCV puro PLC d PH PLC-δ PH domain Dr. M. Engelke
pMSCV puro Raf PA SLP65-∆N
Raf1 391-426, SLP65-∆N
pMSCV puro SCOC CC SLP65-∆N CCit
SCOC 78-159, SLP65-∆N, C-terminal Citrine
pMSCV puro SCOC CC SLP65-∆N-M2,3 CCit
SCOC 78-159, SLP65-∆N-M2,3, C-terminal Citrine
pMSCV puro SCOC CC Tetramer
SCOC 78-159 N125L/N132V
pMSCV puro SCOC CC Tetramer SLP65-∆N-M2,3
SCOC 78-159 N125L/N132V, SLP65-∆N, C-terminal Citrine pMSCV puro SCOC CC
Trimer SLP65-∆N-M2,3
SCOC 78-159 E93V/K97L, SLP65-∆N-M2,3, C-terminal Citrine
pMSCV puro SCOC CC Trimer
SCOC 78-159 E93V/K97L
pMSCV puro SCOC CC SCOC 78-159
pMSCV puro SLP65-∆N CCit
SLP65-∆N, C-terminal Citrine tag
pMSCV puro
SLP65-∆N-M2,3 CCit
SLP65-∆N-M2,3, C-terminal Citrine tag
pMSCV puro slp65-M2,3 CCit
SLP65-M2,3, C-terminal Citrine tag
pMSCV puro SLP65-wt CCit SLP65-wt, C-terminal Citrine tag
pMSCV puro Syk tSH2 Syk tSH2 domains Dr. M. Engelke
pMSCV puro SyktSH2 ∆N CCit
Syk tSH2 domains, SLP65-∆N, C-terminal Citrine tag
pMSCV puro SyktSH2-M2,3 CCit
Syk tSH2 domains,
SLP65-∆N-M2,3, C-terminal Citrine tag
pMSCV puro VN Venus 1-173 Dr. M. Engelke
pMSCV puro VN SLP65-∆N RL
Venus 1-173, SLP65-∆N R372L
pMSCV puro VN SLP65-∆N Venus 1-173, SLP65-∆N pMSCV puro VN
SLP65-∆N-M2,3
Venus 1-173, SLP65-∆N-M2,3
3.1.11 Bacteria
Tab. 3.12: Bacterial strains used in this study
Strain Manufacturer
One Shot TOP10F’ chemo-competentE. coli Life technologies One Shot BL21 (DE3) chemo-competentE. coli Life technologies
3.1.12 Eukaryotic cell lines DT40 (ATCC CRL-2111)
DT40 is a chicken B cell line which derived from an avian leucosis virus-induced bursal lymphoma [6].
This cell line displays IgM on the cell surface and shows a high ratio of targeted to random DNA inte- gration. This facilitates genetic modification via homologous recombination [194].
Ramos(DSMZ: ACC 603)
The Ramos B cell line is established from the ascitic fluid of a 3-year-old boy with Burkitt lymphoma in 1972 [86]. The cell line displays IgM paired with a λ light chain on its surface. It is Epstein-Barr virus-negative.
Platinum-E
Platinum-E is a HEK 293T-derived retroviral packaging cell line [114]. It expresses the structural genes gag,pol andenv of the moloney murine leukemia virus. This allows packaging of transfected DNA into retroviral particles.
3.2 Methods
3.2.1 Genetic methods
Molecular cloning techniques were performed according to established standard protocols as described in Sambrook and Russell [152] if not indicated otherwise.
3.2.1.1 Polymerase chain reaction (PCR)
PCR, originally described in Mullis et al. [115], was used to amplify DNA fragments for following cloning procedures. The enzyme Phusion High-Fidelity polymerase (NEB) was used to catalyze the PCR. The reaction mixture was composed according to the instructions given in the manufacturer’s manual. A 20µl reaction mixture contained 4µl Phusion HF reaction buffer, dNTPs (0.2 mM), DNA oligonucleotides as primers (1µM of each primer), 25-100 ng DNA template and 0.4 U Phusion High- Fidelity DNA polymerase. The indicated standard temperature protocol was used (Table 3.13). For GC-rich templates, the annealing temperature was increased to 60-65.
Tab. 3.13: Temperature gradient used for PCR
Step Temperature [] Time Number of cycles
Initial denaturation 98 2 min 1
Denaturation 98 15 s 30-32
Annealing 55 30 s 30-32
Elongation 72 20 s/kB 30-32
Final elongation 72 10 min 1
3.2.1.2 Overlap extension PCR
Overlap extension PCR, a PCR variant to fuse two DNA fragments [68], was used to create DNA constructs encoding for chimeric or tagged proteins. First, two fragments were amplified by PCR as
fragments. After their isolation by agarose gel electrophoresis and following DNA purification, the two fragments were mixed and served as primers for each other in a following PCR of 15 cycles. Subsequently, primers corresponding to beginning and end of the final product were added at a concentration of 1µM and a PCR of 20 cycles was launched. The annealing temperature of this final PCR was set to 60to increase the specificity.
3.2.1.3 Site directed mutagenesis
The plasmid pCR2.1, which is optimized for cloning procedures, was used for mutagenesis reactions. The protocol was based on the QuikChange method (Stratagene) with modifications according to Edelheitet al. [36]. Two standard PCRs were carried out, one with a forward primer carrying the desired mutation, the other with the complementary reverse primer. The elongation time was chosen to allow a complete amplification of the plasmid. In the end, both reaction mixtures were combined and de- and re-natured according to the indicated temperature protocol (Table 3.14). The reaction mixture was digested with DpnI and transformed into theE. coli strain Top10F’ (see 3.2.1.8)
Tab. 3.14: Temperature gradient for plasmid de- and re-naturation after mutagenesis PCR Temperature [] Time
95 5 min
90 1 min
80 1 min
70 30 s
60 30 s
50 30 s
40 30 s
37 5 min
3.2.1.4 Enzymatic digest of DNA
For following cloning procedures, DNA originating from PCRs or Plasmids was digested using type II restriction enzymes (NEB). Therefore, approx. 1µg DNA was mixed with 0.5µl of each restriction enzyme, the NEB buffer and BSA (final concentration 0.1µg/µL). Water was added to a final reaction volume of 25µL. The digest was performed for 2-16 h.
3.2.1.5 Agarose gel electrophoresis
To separate DNA fragments according to their size, agarose gel electrophoresis was used. 1% or 2%
agarose gels were prepared in TAE buffer, 1µg/mL ethidium bromide was added. The DNA containing samples were mixed with DNA loading buffer and loaded on the agarose gel. To estimate the DNA frag-
